CN107884377A - A kind of application based on cell excretion body nanometer aggregate probe and its in cell-targeting fluorescence and magnetic multi-modality imaging preparation is prepared - Google Patents
A kind of application based on cell excretion body nanometer aggregate probe and its in cell-targeting fluorescence and magnetic multi-modality imaging preparation is prepared Download PDFInfo
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- CN107884377A CN107884377A CN201711190007.4A CN201711190007A CN107884377A CN 107884377 A CN107884377 A CN 107884377A CN 201711190007 A CN201711190007 A CN 201711190007A CN 107884377 A CN107884377 A CN 107884377A
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention discloses a kind of application based on cell excretion body nanometer aggregate probe and its in cell-targeting fluorescence and magnetic multi-modality imaging preparation is prepared, the probe includes the combination probe of metalfluorescent nanometer aggregate probe or magnetic Nano aggregate probe or both；The probe is using metal ion as predecessor, and using the excretion body of cell as carrier carrier, formation is based on cell excretion body nanometer aggregate probe.The present invention's has good chemistry, magnetically or optically stability and biocompatibility based on cell excretion body nanometer aggregate probe, optionally portion's formation in the cell can have the nanometer aggregate probe of fluorescence and magnetic and then mark cell excretion body.By the way that fluorescence and the multifunctional nano aggregate probe of magnetic can be generated to the intracellular associated metal predecessor that introduces; simultaneously; excretion body of the cell-targeting fluorescence imaging preparation except can effectively mark cell made of plasma selenium is added, normal cell can be protected with inducing cell apoptosis.
Description
Technical field
The invention belongs to bioanalysis detection technique field, and in particular to one kind based on cell excretion body nanometer aggregate probe and
Its application in cell-targeting fluorescence and magnetic multi-modality imaging preparation is prepared.
Background technology
At present, cancer is still to be only second to the major causes of death of angiocardiopathy.In various types of cancers, lung cancer
Death rate highest, next to that the carcinoma of the rectum and stomach cancer.In 2015, stomach cancer was listed in the 5th big cancer, caused 81.9 ten thousand people dead
Die.Research is found, if can be diagnosed before cancer metastasis to vitals, what the survival rate of patient may be significantly
Improve.Therefore, the treatment for cancer is early diagnosed to have great importance.Up to the present, a variety of bio-imaging technologies have been
The detection of cancer is applied to, mainly including computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, fluorescence resonance energy
Measure transfer, positron emission tomography image (PET), optical imagery and biological are imaged etc..
Recent decades, nano material are illustrated good by gradually applied to domain variabilities such as bio-imaging and target drug-carryings
Prospect.Nevertheless, nano material is still a unavoidable problem for the toxic action of organ.It is well known that cell
Between tissue by nearby secreting, paracrine and endocrine mutually exchanged, it is in this process, micro- secreted by cell
Nanometer film combination, i.e. Vesicles play an important role.Vesicles below 100 nanometers are referred to as excretion body, it
It is as caused by the entoderm of intracellular body cavity.The composition of Vesicles depends primarily on the type and environment of cell.It is reported that
Various organelles including mitochondria, mRNA, RNA, protein, virus have Vesicles.Vesicles can lead to
Cross and swallow, receive drug therapy and be directly fused in target cell film to transport goods.Therefore, in modem therapies, they
Effect be very important.In addition, the protein and nucleic acid on excretion body have specificity and selectivity, can be used for loading
Specific material.
The content of the invention
Goal of the invention：The problem of existing for prior art, the present invention provide and are based on cell excretion body nanometer aggregate probe, should
Probe good biocompatibility, fluorescent stabilization, it can facilitate and accurate quickly fluorescence labeling excretion body related to detecting.
The present invention also provides based on cell excretion body nanometer aggregate probe and its is preparing cell-targeting fluorescence and magnetic multimode
Application in state imaging agent.
Technical scheme：To achieve these goals, as described herein based on cell excretion body nanometer aggregate probe, including gold
Belong to the combination probe of fluorescence nano aggregate probe or magnetic Nano aggregate probe or both；The nanometer aggregate probe includes metalfluorescent
The combination probe of nanometer aggregate probe or magnetic Nano aggregate probe or both；The metalfluorescent nanometer aggregate probe is with metal ion
As metal ion predecessor, using the excretion body of cell as carrier carrier, the metal nano based on cell excretion body is formed
Cluster fluorescence probe；The magnetic Nano aggregate probe is used as fortune using iron ion as metal ion predecessor, using the excretion body of cell
Defeated body carrier, form the metal magnetic nanometer aggregate probe based on cell excretion body；The combination probe is with iron ion and other gold
Belong to ion population as metal ion predecessor, using the excretion body of cell as carrier carrier, formation is based on cell excretion body
Metalfluorescent nano-cluster and magnetic Nano aggregate probe.
Wherein, other described metal ions are heavy metal ion.
The metal ion be gold ion, silver ion, platinum ion, copper ion, vanadium ion or zinc ion in any one or
Two kinds.
Metalfluorescent nano-cluster and magnetic Nano aggregate probe based on cell excretion body, typically from iron ion and other gold
Any one combination is used as metal front in category ion such as gold ion, silver ion, platinum ion, copper ion, vanadium ion or zinc ion
Thing.It is preferred that using gold and iron ion.Wherein the concentration of gold ion is 10-100umol/L, and the concentration of iron ion is 10-
100umol/L, preferably gold ion concentration are 50umol/L, and the concentration of iron ion is 50umol/L.
Wherein, the excretion body of the cell is the excretion body or angiocardiopathy cell excretion body of tumour cell.The spy
Pin good biocompatibility, fluorescent stabilization, excretion body or angiocardiopathy cell excretion body to tumour cell can be carried out
Fluorescence labeling and detection；Especially there is good selectivity to stomach cancer cell, being capable of selected marker stomach cancer cell well
Excretion body.
Wherein, the nano-cluster fluorescence probe excitation wavelength is 420 or 480nm, and launch wavelength is 580nm or 680nm, and
It will not be changed according to the change of excitation wavelength.
It is of the present invention based on cell excretion body nanometer aggregate probe prepare cell-targeting fluorescence and magnetic it is multi-modal into
As the application in preparation.
Wherein, cell-targeting fluorescence and magnetic the multi-modality imaging preparation includes receiving based on cell excretion body metalfluorescent
Rice aggregate probe and magnetic Nano aggregate probe and plasma selenium.Wherein the concentration of plasma selenium is 10-50umol/L, preferably plasma selenium
Concentration is 30umol/L.
The application of cell-targeting fluorescence and magnetic multi-modality imaging preparation of the present invention in excretion body fluorescence imaging.
Cell-targeting fluorescence and magnetic multi-modality imaging preparation are mainly the excretion body fluorescence imaging applied to cell, can be with
The effective excretion body for detecting excretion body, especially tumour cell；Pass through cell-targeting fluorescence for tumour cell excretion body
Imaging agent can effectively fluorescence imaging, apoptosis in gastric cancer can be even induced while imaging, while protect normal
Cell.
The present invention is by based on cell excretion body nanometer aggregate probe or cell-targeting fluorescence and magnetic multi-modality imaging system
Agent can directly detect the excretion body of mark in the cell in excretion body fluorescence imaging.
The excretion body that directly detection marks in the cell, concretely comprises the following steps and comprises the following steps：By cell and metal ion
After 24 hours, low-speed centrifugal is separated and collected thin for predecessor or cell and metal ion predecessor and plasma selenium co-incubation
Born of the same parents are simultaneously washed three times with phosphate buffer solution, pass through confocal microscope or the high micro- sem observation of intension confocal fluorescent.
The excretion body of probe of the present invention mark, can separation detection by the following method：By cell and metal ion forerunner
After 24 hours, low-speed centrifugal discards cell for thing and plasma selenium co-incubation, and then high speed centrifugation collects supernatant and filter membrane again
It is filtered to remove impurity, excretion body is obtained in last ultrahigh speed low-temperature centrifugation, by being seen under transmission electron microscope
Examine.
The present invention the metal nano aggregate probe based on cell excretion body using golden iron ion as predecessor, the metal such as gold from
Son is reduced generation metalfluorescent nanometer aggregate probe, and iron ion generates oxidation with the ROS Free Radicals in cell excretion body
Ferromagnetic nano probe, the nano-cluster with fluorescent characteristic is generated and loaded so as to cell using the excretion body of tumour cell
Excretion body carries out in situ, real-time mark.The present invention utilizes transmission electron microscope, fourier-transform infrared, x-ray photoelectron spectroscopy
The excretion body extracted, which is characterized, can substantially observe the formation of metal nanometre cluster.In addition, by drawing into the cell
It is multi-modal to enter cell-targeting fluorescence and magnetic made of metalfluorescent nanometer aggregate probe and magnetic Nano aggregate probe and plasma selenium
Imaging agent can effectively mark the excretion body of cell, not only can be for angiocardiopathy cell or tumour cell such as stomach
Cancer cell marker is imaged, and can also be suppressed the propagation of angiocardiopathy cell or tumour cell and be protected normal cell.Pass through
Test is carried out to expressing gene to find after metalfluorescent nanometer aggregate probe and magnetic Nano aggregate probe and plasma selenium is introduced
Intracellular metal combination gene and tumour eliminate gene and obvious up-regulation occur.
Using tumour cell in the present invention tests, such as the carry out excretion body mark of stomach cancer cell and detection, to it
The excretion body of his cell is equally possible effectively.
Beneficial effect：Compared with prior art the invention has the advantages that：
Being had based on cell excretion body nanometer aggregate probe for the present invention is good with well chemical, magnetically or optically steady
Qualitative and biocompatibility, the excretion body nanometer aggregate probe by the use of cell excretion body as transport carrier, by thin
Intracellular introduces relevant metal ions predecessor, optionally nano-cluster of the portion's formation in the cell with fluorescence and magnetic can visit
Pin and then mark cell excretion body.By introducing metalfluorescent nano-cluster and magnetic Nano aggregate probe and plasma selenium to intracellular
The excretion body of manufactured cell-targeting fluorescence and magnetic multi-modality imaging preparation except can effectively mark cell, can also enter
One step ground inducing cell apoptosis, protects normal cell.Introducing metalfluorescent is can be found that by detecting intracellular gene expression
Nano-cluster and magnetic Nano aggregate probe and plasma selenium can cause metal combination gene and tumour to eliminate the up-regulation of gene.
Nano-cluster probe preparation method of the invention based on cell excretion body is simple and convenient, and raw material sources are extensive；Simultaneously should
The detection that probe is used for cell excretion body is simple and easy to do, quick, and intuitively, toxicity is low, high sensitivity.
Brief description of the drawings
Fig. 1 is the transmission electron microscope image for the cell excretion body that the present invention marks；
The metal nano aggregate probe that Fig. 2 is the present invention is used for the stomach cancer cell fluorescence co-focusing micrograph in situ from imaging；
The metal nano aggregate probe that Fig. 3 is the present invention shows for the stomach cancer cell high intension confocal fluorescent in situ from imaging
Micro mirror figure；
Fig. 4 is that the metal nano aggregate probe of the present invention is used for the fluorogram of living animal imaging；
Fig. 5 is the fluorescence pattern and particle diameter distribution graph of a relation of the metal nano aggregate probe of the present invention；
Fig. 6 is situation of change relation of the stomach cancer cell in every expressing gene after the processing of metal nano aggregate probe
Figure.
Embodiment
The present invention will be further described with reference to the accompanying drawings and examples.
Embodiment 1
Cell (SGC-7901, Hela, HepG2 and L02) culture is reached to 90% coverage, Ran Houfen in 6 orifice plates
Not Jia Ru 50umol/L gold chloride 5ul, 50umol/L solution of ferrous chloride 5ul, cell and metal ion predecessor are trained jointly
After supporting 24 hours and after 48 hours, centrifugation in 20 minutes is centrifuged under 2000g respectively and discards cell, then centrifuges and receives under 10000g
Collection supernatant is simultaneously filtered to remove impurity with 0.22 micron of filter membrane to it, finally with temperature of the 120000 g centrifugal force at 4 DEG C
The lower centrifugation of degree obtains excretion body in 70 minutes, and its pattern is observed under JEOL2100 transmission electron microscopes, as a result such as Fig. 1 institutes
Show.One layer of golden iron nano-cluster, the wherein particle diameter of gold nanoclusters have been wrapped up around 40 nanometers of excretion body as can see from Figure 1
About 3 nanometers, the particle diameter of iron nano-cluster is about 5 nanometers.
Embodiment 2
The difference identical with the method for embodiment 1 of embodiment 2 is, gold ion and iron ion are substituted for into 10umol/L platinum
Ion and 10umol/L copper ions, it is made based on cell excretion body platinum, copper fluorescence nano aggregate probe.
Embodiment 3
The difference identical with the method for embodiment 1 of embodiment 2 is, gold ion and iron ion are substituted for into 100umol/L
Silver ion and 100umol/L zinc ions, it is made based on cell excretion body silver, zinc fluorescence nano aggregate probe.
Embodiment 4
The difference identical with the method for embodiment 1 of embodiment 2 is, gold ion and iron ion are substituted for into 100umol/L
Iron ion, it is made based on cell excretion body iron oxide magnetic nano probe.
Embodiment 5
SGC-7901, Hela, HepG2 and L02 cell line are respectively placed in containing the hyclone of volume fraction 10%
In DMEM (the μ g/mL penicillin 100IU/mL of streptomysin 100) culture medium, in 37 DEG C, contain 5%CO2, the incubator of 95% humidity
Middle culture.Before imaging, cell is put into and cultivates the coverage for reaching 90% in 6 orifice plates, is separately added into 50umol/L gold chlorides
5ul, 50umol/L solution of ferrous chloride 5ul, co-incubation are separated with 3000 turns of speed after 24 hours and collect cell and be used in combination
Phosphate buffer solution washs three times, carries out confocal microscope imaging, as a result as shown in Figure 2.Profit is real as can be seen from Figure 2
Test result to show, by the labeled SGC-7901 cells of gold, iron nanometer aggregate probe, it can be seen that there is obvious fluorescence in cytoplasm
Enhancing, illustrate the metalfluorescent nano-cluster and magnetic Nano aggregate probe of the present invention, such as gold, iron nanometer aggregate probe can be optionally
Mark the horizontal change of stomach cancer cell simultaneous intracellular free radicals.
Identical method equally is used, is separately added into 50umol/L gold chloride 5ul, 50umol/L solution of ferrous chloride 5ul
And 30umol/L sodium selenite 5ul, co-incubation are separated with 3000 turns of speed after 24 hours and collect cell and use phosphoric acid
Cushioning liquid washs three times, carries out confocal microscope imaging, as a result as shown in Figure 2.Profit experiment knot as can be seen from Figure 2
Fruit shows, by introducing cell-targeting made of metalfluorescent nano-cluster and magnetic Nano aggregate probe and plasma selenium to intracellular
Fluorescence and magnetic multi-modality imaging preparation can effectively mark the excretion body of cell, optionally can mark outside stomach cancer cell
The horizontal change of body simultaneous intracellular free radicals is secreted, and effect is more preferable.
Embodiment 6
SGC-7901, Hela, HepG2 and L02 cell line are respectively placed in containing the hyclone of volume fraction 10%
In DMEM (the μ g/mL penicillin 100IU/mL of streptomysin 100) culture medium, in 37 DEG C, contain 5%CO2, the incubator of 95% humidity
Middle culture.Before imaging, cell is put into and cultivates the coverage for reaching 90% in 6 orifice plates, is separately added into 50umol/L gold chlorides
5ul, 50umol/L solution of ferrous chloride 5ul, co-incubation 24 hours, separated with 3000 turns of speed and collect cell and use phosphorus
Acid buffering solution washs three times, and the situation under high intension imaging system in Continuous Observation culture 24 hours, while records glimmering
Luminous intensity, experimental result are as shown in Figure 3.SGC-7901, Hela, HepG2 and L02 cell are adding fluorescence as can be seen from Figure 3
Fluorescence intensity change after probe between 24 hours, the fluorescence intensity of cell constantly strengthens over time in whole process, card
Bright metalfluorescent nano-cluster and magnetic Nano aggregate probe based on cell excretion body have good selectivity.
Embodiment 7
Experiment made on the living carries out (offer of health science portion of Peking University, average weight 18-20 on 3 weeks big nude mices
Gram).It is 5x 10 by concentration7mL-1SCG-7901 tumour cells FBS suspension be subcutaneously injected on the outside of the chest of nude mice.4 weeks
Afterwards, obvious tumour can be formd.Nude mice is divided into experimental group and control group, every group of 3 mouse.By tail vein injection to
Nude mice injects 100umol/L gold chloride 2ul, 100umol/L solution of ferrous chloride 2ul and 50umol/L sodium selenite 1ul, comments
The ability of its oneself imaging of valency 0-48h, and living body fluorescent image is shot at regular intervals, as a result as shown in Figure 4.Can be with from figure
Find out after injection of metallic predecessor and plasma selenium, the fluorescence at mouse tumor position gradually strengthens, and reaches at the 24th hour
Maximum, by isolated organ imaging it can be found that fluorescence is focused primarily upon in tumour, it was demonstrated that the tool of the probe combination plasma selenium
There is good targeting.
Embodiment 8
Cell (SGC-7901, Hela, HepG2 and L02) culture is reached to 90% coverage in 6 orifice plates, then will
50umol/L gold chloride 5ul, 50umol/L solution of ferrous chloride 5ul and 30umol/L sodium selenite 5ul are separately added into, are added
Into culture medium with inducing cell fabricated in situ nano-cluster.After culture 24 hours, separate and collect with 3000 turns of speed
Cell is simultaneously washed three times with phosphate buffer solution.Then the nano-cluster of biology in situ synthesis is obtained using freeze-thaw method.
Take gold nanoclusters corresponding the solvent such as ultra-pure water or phosphate buffer solution of a certain amount of biology in situ synthesis
Dilute and be added in clean 4mL quartz colorimetric utensils, measured with 420nm excitation wavelengths, as a result as shown in Figure 5A.Can be with from Fig. 5 A
Find out the fluorescence for launching 580nm under the exciting of 420nm wavelength from the nano-cluster extracted into the cell, in addition excitation wavelength 480nm
When, launch wavelength 680nm, and will not be changed according to the change of excitation wavelength.
The nano-cluster of intracellular fabricated in situ is extracted with freeze-thaw method, is scattered in after centrifugation in ethanol.
By hanging drop cleaning copper mesh on, it is to be dried after under JEOL2100 transmission electron microscopes observe nano-cluster pattern with
And particle diameter.As a result as shown in Figure 5 B.The particle diameter distribution of nano-cluster is homogeneous as can be seen from Figure 5B, is concentrated mainly on 2.3 nanometers of left sides
It is right.
Embodiment 9
SGC-7901 and L02 cell culture is reached in 6 orifice plates to 90% coverage, is separately added into 50umol/L chlorine gold
Sour 5ul, 50umol/L solution of ferrous chloride 5ul and 30umol/L sodium selenite 5ul, co-incubation is after 24 hours, with 3000
Turn speed separate and collects cell and washed three times with phosphate buffer solution, then with PBS withReagent carries out clear
Wash to separate RNA, and be stored in subzero 196 DEG C of environment to carry out further experiment.SGC-7901 is tested using microarray
Difference between L02 cell mRNAs, experimental result are as shown in Figure 6.From Fig. 6 results, stomach cancer cell is visited by fluorescence
Every expressing gene after pin processing changes, and the expression of wherein metal combination gene and cell ablation gene occurs bright
Aobvious up-regulation, it was demonstrated that metalfluorescent nano-cluster and magnetic Nano aggregate probe and plasma selenium based on cell excretion body can lure
Cancer cell is further killed on the basis of guided cell fluorescence imaging.
Claims (10)
1. one kind is based on cell excretion body nanometer aggregate probe, it is characterised in that the nanometer aggregate probe includes metalfluorescent nanometer
The combination probe of aggregate probe or magnetic Nano aggregate probe or both；The metalfluorescent nanometer aggregate probe using metal ion as
Metal ion predecessor, using the excretion body of cell as carrier carrier, it is glimmering to form the metal nanometre cluster based on cell excretion body
Light probe；The magnetic Nano aggregate probe is used as carrier using iron ion as metal ion predecessor, using the excretion body of cell
Carrier, form the metal magnetic nanometer aggregate probe based on cell excretion body；The combination probe with iron ion and other metals from
Sub-portfolio, using the excretion body of cell as carrier carrier, forms the gold based on cell excretion body as metal ion predecessor
Belong to fluorescence nano cluster and magnetic Nano aggregate probe.
2. according to claim 1 be based on cell excretion body nanometer aggregate probe, it is characterised in that the metal ion is attached most importance to
Metal ion.
3. according to claim 1 or 2 be based on cell excretion body nanometer aggregate probe, it is characterised in that the metal ion
For any one in gold ion, silver ion, platinum ion, copper ion, vanadium ion or zinc ion or two kinds.
4. according to claim 1 be based on cell excretion body nanometer aggregate probe, it is characterised in that the iron ion and other
Metal ion is combined as iron ion and any one group in gold ion, silver ion, platinum ion, copper ion, vanadium ion or zinc ion
Close.
5. according to claim 1 be based on cell excretion body nanometer aggregate probe, it is characterised in that the excretion body of the cell
Excretion body or angiocardiopathy cell excretion body for tumour cell.
6. cell excretion body according to claim 1 is based on cell excretion body nanometer aggregate probe, it is characterised in that described to receive
Rice aggregate probe fluorescence exciting wavelength is 420nm or 480nm, and launch wavelength is 580nm or 680nm.
7. cell-targeting fluorescence and magnetic multimode are being prepared based on cell excretion body nanometer aggregate probe described in a kind of claim 1
Application in state imaging agent.
8. application according to claim 7, it is characterised in that the cell-targeting fluorescence and magnetic multi-modality imaging preparation
Including being based on cell excretion body metalfluorescent nanometer aggregate probe and magnetic Nano aggregate probe and plasma selenium based on cell excretion body.
9. the cell-targeting fluorescence and magnetic multi-modality imaging preparation described in a kind of claim 7 are in excretion body fluorescence imaging
Using.
10. application according to claim 9, it is characterised in that the cell-targeting fluorescence and magnetic multi-modality imaging system
Agent comprises the following steps in excretion body fluorescence imaging：By cell and metal ion predecessor and plasma selenium co-incubation 24 hours
Afterwards, low-speed centrifugal separates and collects cell and washed three times with phosphate buffer solution, by confocal microscope or height
Contain the micro- sem observation of confocal fluorescent.
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Cited By (3)
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| CN111235216A (en) * | 2020-02-26 | 2020-06-05 | 东南大学 | Method for accurately detecting and killing pathogenic microorganisms by using intelligent bioprobe |
| CN111909900A (en) * | 2020-07-19 | 2020-11-10 | 东南大学 | Method for enhancing immune response based on in-situ self-assembly intelligent nanoparticles |
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| CN103083687A (en) * | 2013-01-16 | 2013-05-08 | 东南大学 | Application of silver and platinum nano-cluster in tumor targeted imaging |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109432427A (en) * | 2018-12-25 | 2019-03-08 | 上海纳米技术及应用国家工程研究中心有限公司 | Using excretion body as the preparation method and products thereof of the cancer target thermotherapy material of carrier |
| CN111235216A (en) * | 2020-02-26 | 2020-06-05 | 东南大学 | Method for accurately detecting and killing pathogenic microorganisms by using intelligent bioprobe |
| CN111909900A (en) * | 2020-07-19 | 2020-11-10 | 东南大学 | Method for enhancing immune response based on in-situ self-assembly intelligent nanoparticles |
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